1. Antibody Structure
Antibody molecules typically are Y-shaped molecules whose basic unit consist of four polypeptides, two identical heavy chains and two identical light chains, which are covalently linked together by disulfide bonds. Each of these chains is folded in discrete domains. The C-terminal regions of both heavy and light chains are conserved in sequence and are called the constant regions, also known as C-domains. The N-terminal regions, also known as V-domains, are variable in sequence and are responsible for the antibody specificity. The antibody specifically recognizes and binds to an antigen mainly through six short complementarity-determining regions located in their V-domains (see FIG. 1).
In this specification abbreviations are used having the following meaning.
C-domain: Constant domain PA1 V-domain: Variable domain PA1 V.sub. : Variable domain of the light chain PA1 V.sub.H : Variable domain of the heavy chain PA1 Fv: dual chain antibody fragment containing both a V.sub.H and a V.sub.L PA1 scFv: single-chain Fv (V.sub.H and V.sub.L genetically linked either directly or via a peptide linker) PA1 CDR: Complementarity-determining region PA1 ELISA: Enzyme Linked Immuno Sorbent Assay PA1 PCR: Polymerase Chain Reaction PA1 IPTG: IsoPropyl-.beta.-ThioGalactopyranoside PA1 PBS: Phosphate Buffered Saline PA1 PBST: Phosphate Buffered Saline with 0.15% Tween PA1 TMB: 3,3',5,5'-TetraMethylBenzidine
It is generally known that proteolytic digestion of an antibody with papain yields three fragments. The fragment containing the CH.sub.2 and CH.sub.3 domains of the two heavy chains connected by the complete hinge (see FIG. 1) crystallises very easily and was therefore called Fc fragment. The two other fragments are identical and were called Fab fragments, as they contained the antigen-binding site. Digestion with pepsin is such that the two Fab's remain connected via the hinge, forming only two fragments: Fc' and Fab.sub.2.
The Fv is the smallest unit of an antibody which still contains the complete binding site (see FIG. 1) and full antigen binding activity. It consists of only the V-domains of the heavy and light chains thus forming a small, heterodimeric variable fragment or Fv. Fv's have a molecular weight of about 25 kD, which is only one sixth of the parent whole antibody (in the case of an IgG). Previously Fv's were only available by proteolysis in a select number of cases (Givol, 1991). The production of Fv's can now be achieved more routinely using genetic engineering methods through cloning and expressing DNA encoding only the V-domains of the antibody of interest. Smaller fragments, such as individual V-domains (Domain Antibodies or dABs, Ward et al., 1989), and even individual CDR's (Williams et al., 1989; Taub et al., 1989) were shown to retain the binding characteristics of the parent antibody. However, this is not achievable on a routine basis: most naturally occurring antibodies need both a V.sub.H and a V.sub.L to retain full immunoreactivity. For example, in the case of V.sub.H D1.3 (Ward et al., 1989), although it still binds hen egg lysozyme (HEL) with an affinity close to that of the parent antibody, it was shown that loss of specificity was observed in that it can no longer distinguish turkey lysozyme from HEL, whereas the Fv can (Berry and Davies, 1992). Although murine dABs can be obtained more routinely from spleen libraries (Ward et al., 1989), the approach is unsustainable because of the many problems associated with their production and physical behaviour: expression is extremely poor, affinity tends to be low, stability and solubility in water is low, and non-specific binding is usually very high. According to the literature a possible explanation of these undesirable characteristics is the exposure of the hydrophobic residues which are normally buried in the V.sub.H -V.sub.L interface. The exposed hydrophobic patches are thought to contribute to aggregation of the protein inside the cells and/or in the culture medium, leading to poor expression and/or poor solubility (Anthony et al., 1992; Ward et al., 1989). The hydrophobic patches can also explain the high non-specific binding described by Berry and Davies, 1992. These problems clearly limit the usefulness of these molecules. Most of the Camelid antibodies appear to be an exception to this rule in that they only need one V-domain, namely V.sub.H, to specifically and effectively bind an antigen (Hamers-Castermans et al., 1993). In addition, preliminary data indicate that they seem not to suffer from the disadvantages of mouse dABs, as these camelid antibodies or fragments thereof are soluble and have been shown to express well in yeast and Aspergillus moulds. These observations can have important consequences for the production and exploitation of antibody-based products, see patent application WO 94/25591 (UNILEVER et al., first priority date Apr. 29, 1993).
2. Production of Antibody Fragments
Several microbial expression systems have already been developed for producing active antibody fragments, e.g. the production of Fab in various hosts, such as E. coli (Better et al., 1988, Skerra and Pluckthun, 1988, Carter et al., 1992), yeast (Horwitz et al., 1988), and the filamentous fungus Trichoderma reesei (Nyyssonen et al., 1993) has been described. The recombinant protein yields in these alternative systems can be relatively high (1-2 g/l for Fab secreted to the periplasmic space of E. coli in high cell density fermentation, see Carter et al., 1992), or at a lower level, e.g. about 0.1 mg/l for Fab in yeast in fermenters (Horwitz et al., 1988), and 150 mg/l for a fusion protein CBHI-Fab and 1 mg/l for Fab in Trichoderma in fermenters (Nyyssonen et al., 1993) and such production is very cheap compared to whole antibody production in mammalian cells (hybridoma, myeloma, CHO). Although the latter can give yields of the order of 1 g/l in high cell density fermentation, it is a time-consuming and very expensive manufacturing method resulting in a cost price of about 1000 .English Pound./gram of antibody. It was further demonstrated that plants can be used as hosts for the production of both whole antibodies (Hiatt et al., 1989) and scFv's (Owen et al., 1992, Firek et al., 1993), whereby yields of up to 0.5% of the total soluble protein content in tobacco leaves were mentioned.
The fragments can be produced as Fab's or as Fv's, but additionally it has been shown that a V.sub.H and a V.sub.L can be genetically linked in either order by a flexible polypeptide linker, which combination is known as an scFv (Bird et al. (1988), Huston et al. (1988), and granted patent EP-B-0281604 (GENEX/ENZON LABS INC.; first priority date Sep. 2, 1986).
3. Bivalent and Bispecific Antibodies and Antibody Fragments
The antibody fragments Fab, Fv and scFv differ from whole antibodies in that the antibody fragments carry only a single antigen-binding site. Recombinant fragments with two binding sites have been made in several ways, for example, by chemical cross-linking of cysteine residues introduced at the C-terminus of the V.sub.H of an Fv (Cumber et al., 1992), or at the C-terminus of the V.sub.L of an scFv (Pack and Pluckthun, 1992), or through the hinge cysteine residues of Fab's (Carter et al., 1992). Another approach to produce bivalent antibody fragments is described by Kostelny et al. (1992) and Pack and Pluckthun (1992) and is based on the inclusion of a C-terminal peptide that promotes dimerization.
When two different specificities are desired, one can generate bispecific antibody fragments. The traditional approach to generate bispecific whole antibodies was to fuse two hybridoma cell lines each producing an antibody having the desired specificity. Because of the random association of immunoglobulin heavy and light chains, these hybrid hybridomas produce a mixture of up to 10 different heavy and light chain combinations, only one of which is the bispecific antibody (Milstein and Cuello, 1983). Therefore, these bispecific antibodies have to be purified with cumbersome procedures, which considerably decrease the yield of the desired product.
Alternative approaches include in-vitro linking of two antigen specificities by chemical cross-linking of cysteine residues either in the hinge or via a genetically introduced C-terminal Cys as described above. An improvement of such in vitro assembly was achieved by using recombinant fusions of Fab's with peptides that promote formation of heterodimers (Kostelny et al., 1992). However, the yield of bispecific product in these methods is far less than 100%.
A more efficient approach to produce bivalent or bispecific antibody fragments, not involving in vitro chemical assembly steps, was described by Holliger et al. (1993). This approach takes advantage of the observation that scFv's secreted from bacteria are often present as both monomers and dimers. This observation suggested that the V.sub.H and V.sub.L of different chains can pair, thus forming dimers and larger complexes. The dimeric antibody fragments, also named "diabodies" by Hollinger et al., in fact are small bivalent antibody fragments that assembled in vivo. By linking the V.sub.H and V.sub.L of two different antibodies 1 and 2, to form "cross-over" chains V.sub.H 1V.sub.L 2 and V.sub.H 2-V.sub.L 1 (see FIG. 2B), the dimerisation process was shown to reassemble both antigen-binding sites. The affinity of the two binding sites was shown to be equal to the starting scFv's, or even to be 10-fold increased when the polypeptide linker covalently linking V.sub.H and V.sub.L was removed, thus generating two proteins each consisting of a V.sub.H directly and covalently linked to a V.sub.L not pairing with the V.sub.H (see FIG. 2C). This strategy of producing bispecific antibody fragments was also described in several patent applications. Patent application WO 94/09131 (SCOTGEN LTD; priority date Oct. 15, 1992) relates to a bispecific binding protein in which the binding domains are derived from both a V.sub.H and a V.sub.L region either present at two chains or linked in an scFv, whereas other fused antibody domains, e.g. C-terminal constant domains, are used to stabilise the dimeric constructs. Patent application WO 94/13804 (CAMBRIDGE ANTIBODY TECHNOLOGY/MEDICAL RESEARCH COUNCIL; first priority date Dec. 4, 1992) relates to a polypeptide containing a V.sub.H and a V.sub.L which are incapable of associating with each other, whereby the V-domains can be connected with or without a linker.
Mallender and Voss, 1994 (also described in patent application WO 94/13806; DOW CHEMICAL CO; priority date Dec. 11, 1992) reported the in vivo production of a single-chain bispecific antibody fragment in E. coli. The bispecificity of the bivalent protein was based on two previously produced monovalent scFv molecules possessing distinct specificities, being linked together at the genetic level by a flexible polypeptide linker. The thus formed V.sub.H 1-linker-V.sub.L 1-linker-V.sub.H 2-linker-V.sub.L 2 fragment (see FIG. 2A) was shown to contain both antigen binding specificities 1 and 2. (1=anti-fluorescein, 2=anti-single-stranded DNA). Traditionally, whenever single-chain antibody fragments are referred to, a single molecule consisting of one heavy chain linked to one (corresponding) light chain in the presence or absence of a polypeptide linker is implicated. When making bivalent or bispecific antibody fragments through the `diabody` approach (Holliger et al., (1993) and patent application WO 94/09131) or by the `double scFv` approach (Mallender and Voss, 1994 and patent application WO 94/13806), again the V.sub.H is linked to a (the corresponding) V.sub.L.
It is realised that claims 32 and 33 of patent application WO 93/11161 (ENZON INC.; priority date Nov. 25, 1991) and the corresponding passages in that specification on page 22, lines 1-10 may read on a polypeptide comprising two V.sub.L 's fused together via a flexible polypeptide linker, and on a polypeptide comprising two V.sub.H 's fused together via a flexible polypeptide linker, respectively. However, no examples were given to substantiate this approach, thus it was in fact a hypothetical possibility instead of an actually produced compound.
A skilled person would not have expected that such approach would be viable for at least three reasons. Firstly, it is widely recognised that immunoglobulin heavy chains (excluding the above described camel immunoglobulins) have very limited solubility and spontaneously precipitate out of aqueous solution when isolated from their light chain partners. Secondly, several groups have shown (Ward et al., 1989, Berry and Davies, 1992, and Anthony et al., 1992) that expression of V.sub.H 's in the absence of V.sub.L 's is hampered by extremely poor yields of unstable product with many undesirable properties, e.g. non-specific binding. Thirdly in patent application WO 94/13804 it was described on page 31 lines 10-12, that in computer modelling experiments they could not model as heterodimers V.sub.H --V.sub.H and V.sub.L --V.sub.L given the constraints of short linkers.
Thus the simple suggestion given in patent application WO 93/11161 is not an enabling disclosure leading a skilled person to try with a reasonable expectation of success whether such suggestion would work; therefore, that patent application should not be considered as relevant prior art for the present invention.